Deoxy sugars are examples of so-called rare sugars, which are found in small amounts in plant-based materials, such as wood resources, seaweeds and sugar beet and sugar cane. Specific deoxy sugars have been found useful for example for sweetener applications as well as for pharmaceutical and cosmetic applications.
Deoxy sugars are known to exist in L-form and in D-form. For example, fucose exists as L-fucose and D-fucose.
One example of deoxy sugars of special interest is fucose, also named 6-deoxygalactose. Fucose is found in a wide variety of natural products from many different sources, in both D-form and L-form. Interest in L-fucose has recently increased because of its potential in the medical field in treating various disease conditions, such as tumors, inflammatory conditions and disorders relating to the human immune system. L-fucose has also applications in the cosmetic field, for instance as a skin moisturizing agent.
In accordance with Merck Index, Twelfth Edition, 1996, crystalline L-fucose has a melting point of 140° C. and an optical rotation of −75.6°.
L-fucose occurs for instance in several human milk oligosaccharides.
In plant material, fucose is typically associated with plant polysaccharides, which are often highly branched structures having L-fucopyranosyl units either at the ends of or within the polysaccharide chains. In some cases, even methylated fucopyranosyl units occur in plant polysaccharides.
L-fucose or methylated L-fucopyranosyl units occur in the cell walls of potato, cassava tuber and kiwi fruit, in the seed polysaccharides of soybean and in winged bean varieties and canola, for example.
Seaweed polysaccharides, found in the intercellular mucilage, form complex structures and are often composed of sulfated L-fucose polymers, named fucoidan. Seaweeds of particular importance for the extraction of fucoidan are Ecklonia kurome, Laminaria angustata var longissima, Fucus vesiculosus, Kjellmaniella crassifolia, Pelvetia canaliculata and Fucus serratus L.
Furthermore, extracellular polysaccharides from various bacteria, fungi and micro-algae contain L-fucose.
L-fucose can be obtained from natural sources, such as algae by various extraction methods. These raw materials of natural origin used for the recovery of fucose are typically multicomponent mixtures. The separation of fucose with sufficient purity has presented a problem in the state of the art.
L-fucose has been obtained by hydrolysis of fucoidan occurring in Phaeophyceae algae. Black, W. A. P. et al. disclose an optimized fucoidan extraction method in “Manufacture of algal chemicals. IV. Laboratory-scale isolation of fucoidan from brown marine algae”, J. Sci. Food Agric. 3:122–129 (1952). The highest yields were obtained by extraction (pH 2.0–2.5) with hydrochloric acid at a temperature of 70° C. for 1 h. A ratio (w/v) of 1 unit algae to 10 units liquid was shown to be optimal. This procedure yielded about 50% of the total L-fucose. Three subsequently performed acid extractions yielded more than 80% L-fucose. The crude fucoidan was isolated from the acid extraction liquid by neutralization and evaporation to dryness.
U.S. Pat. No. 3,240,775, Kelco Co. (published 15 Mar. 1966) discloses a method of preparing crystals of an α-L-fucoside and L-fucose comprising the steps of heating a mixture of fucoidan, concentrated hydrogen chloride and methanol until the fucoidan is substantially depolymerized and desulfated, and thereafter recovering, from said mixture, a degradation product which consists of methyl α-L-fucoside and, after subsequent hydrolysis, L-fucose.
Example VIII of the above-mentioned reference discloses a process of obtaining crystalline L-fucose from said mixture containing fucoidan degradation products by removing the α-L-fucoside (methyl α-L-fucoside), treating the mixture thus obtained with 1 N sulfuric acid, precipitating sulfuric acid with Ba(OH)2, treating the solution with cation exchange resins (Amberlite IR-120 in H+ form) and activated carbon, concentrating the colorless solution in vacuo to a syrup and diluting the syrup with hot methanol. Ether was added to the diluted solution, and after seeding with L-fucose the mixture was kept refrigerated for 8 to 12 days. Crystalline L-fucose with a melting point of 136 to 138° C. was obtained. In accordance with Example IX, the same procedure provided crystalline L-fucose with a melting point of 136 to 139° C.
Japanese patent publication 63027496 A2 (Takemura, M et al., Towa Chem. Ind.) describes direct extraction of L-fucose from algae belonging to the family of the Chordariaceae or Spermatochnaceae. The algae were dispersed in water and treated with concentrated sulfuric acid. The obtained hydrolyzate was cooled and the algae residues were removed by filtration. The pH of the filtrate was adjusted to 5, the filtrate was treated with charcoal and filtered. A yeast was added to the filtrate to digest the saccharides other than L-fucose. The mixture was treated with charcoal and filtered. The filtrate was subjected to deionization treatment with cation and anion exchange resins and concentrated. The concentrated sugar solution was mixed with ethanol and allowed to crystallize. In this way, L-fucose with a purity of 98.7% was obtained. Melting point data for the L-fucose product was not given.
F. M. Rombouts and J. F. Thibault describe the isolation of pectins from an ethanol-insoluble residue of sugar beet pulp in Carbohydrate Research 1986, 154, pp. 177–187. The isolated pectins were purified by chromatography on DEAE-cellulose or by precipitation with CuSO4. The pectins had relatively high contents of neutral sugars. The main neutral sugars in each pectin were arabinose and galactose; other sugars present were rhamnose, fucose, xylose, mannose and glucose. Fucose was not separated from the sugar/pectin mixture.
V. A. Derevitskaya et al. (Dokl. Akad. Nauk. SSSR (1975), 223(5) 1137–9) describe the separation of complex mixtures of oligosaccharides by anion-exchange chromatography. In accordance with the disclosure, 2-amino-2-deoxyglucitol, glucosamine, galactose and fucose were successfully separated from oligosaccharide mixtures, buffered by 0.2 M borate, by anion-exchange chromatography.
M. H. Simatupang describes ion-exchange chromatography of some neutral monosaccharides and uronic acids in J. Chromatogr. (1979), 178(2), 588–91. The reference discloses ion-exchange chromatography of complex mixtures of uronic acids and monosaccharides containing fucose and mannuronic and guluronic acids utilizing a borate buffer system. The chromatographic system employed a steel column containing HA-X4 or BA-X4 (borate form) anion exchangers and a buffer system of various borate concentrations at various pH values.
D. Balaghova et al. studied the changes of the saccharide portion of maple wood in the course of prehydrolysis in Vybrane Procesy Chem. Spracovani Dreva (1996), 187–192 (Publisher: Technicka Univerzita Zvolen, Zvolen, Slovakia). The main monosaccharides found in maple wood were D-glucose, D-xylose, L-rhamnose, L-fucose, L-arabinose, D-mannose and D-galactose. L-fucose was not separated from the sugar mixture.
L-fucose can also be obtained via chemical synthesis from L-arabinose (Tanimura, A., Synthesis of L-fucose, Chem. Abstr. 55:12306 (1961)), from D-glucose (Chiba, T. & Tejima, S., A new synthesis of α-L-fucose, Chem. Pharm. Bull. 27:2838–2840 (1979)), from methyl-L-rhamnose (Defaye, J., et al., An efficient Synthesis of L-fucose and L-(4-2H)fucose, Carbohydrate Res. 126:165–169 (1984)), from D-mannose (Gesson, J-P et al., A short synthesis of L-fucose and analogs from D-mannose, Tetrahedron Lett. 33:3637–3640 (1992)) and from D-galactose (Dejter-Juszynski, M & Flowers, H-M., Synthesis of L-fucose, Carbohydrate Res. 28:144–146 (1973); Kristen, H., et al., Introduction of a new selective oxidation procedure into carbohydrate chemistry—An efficient conversion of D-galactose into L-fucose, J. Carbohydr. Chem. 7:277–281(1988); Sarbajna, S. et al., A novel synthesis of L-fucose from D-galactose, Carbohydr. Res. 270:93–96 (1965)).
Enzymatic and microbial synthesis has also been used for the production of L-fucose.
C. Wong et al. disclose an enzymatic synthesis of L-fucose and analogs thereof in J. Org. Chem., 60:7360–7363 (1995). L-fucose is produced by enzymatic synthesis from dihydroxyacetone phosphate (DHAP) and DL-lactaldehyde catalyzed by L-fuculose-1-phosphate aldolase, followed by reaction with acid phosphatase and L-fucose isomerase. The L-fucose product was isolated by Dowex 50W-X8 (Ba2+ form) chromatography, optionally combined with separation by silica gel.
EP 102 535, Hoecst AG (published 14 Mar. 1984) discloses a process for the production of deoxysugars selected from fucose and rhamnose by fermentation using the genera Alcaligenes, Klebsiella, Pseudomonas or Enterobacter, which produce extracellular polysaccharides containing more than 10% fucose and/or rhamnose. It is recited that fucose and/or rhamnose are recovered from the hydrolyzate of the fermentation product by chromatography, ion-exchange or adsorption (for example with zeolites) or by further fermentation treatment. In the examples of the EP patent, rhamnose and fucose are recovered by further fermentation treatment.
U.S. Pat. No. 4,772,334. Kureha Kagaku Kogyo Kabushiki Kaisha (published Sep. 20, 1988) discloses a process for producing highly pure rhamnose from gum arabic. The process comprises partial hydrolysis of gum arabic in an aqueous solution of a mineral acid, neutralization and treatment with a polar organic solvent to obtain an aqueous solution containing monosaccharides formed by the hydrolysis of gum arabic, and subjecting the aqueous solution thus obtained to strongly acid cation exchange chromatography and then to a method of adsorption and separation using activated carbon.
WO 02/27039, Xyrofin Oy (published 4 Apr. 2002) discloses a process for recovering a monosaccharide selected from the group consisting of rhamnose, arabinose and mixtures thereof from a solution containing the same by a multistep process comprising at least one step where a weakly acid cation exchange resin is used for the chromatographic separation.
One of the problems associated with known processes is that they provide the desired deoxy sugars as a mixture with other closely related sugars or that they do not provide the deoxy sugars, such as fucose with a sufficient degree of purity. Direct extraction from brown algae is costly, and subject to seasonal variations in the supply volume and quality. On the other hand, the production of L-fucose via chemical synthesis for instance from other sugars may be costly and suffer from low yield. Furthermore, it has been problematical to prepare suitable starting fucose solutions for the crystallization of fucose to obtain a crystalline fucose product having a purity of more than 99%.
Furthermore, the recovery of deoxy sugars from one another has presented a problem in the state of the art due to the closely related structures thereof. In many separation processes, the deoxy sugars behave in the same way, whereby no essential separation between these closely-related sugars occurs. Instead, they are often recovered as an admixture in the same fraction.
It has now been found that fucose and other deoxy sugars with high purity can be effectively recovered from biomass-derived solutions containing deoxy sugars and for example aldose and pentose sugars using a novel chromatographic separation method. It was also found that high purity fucose crystals with a melting point higher than 141° C., preferably higher than 145° C., can be obtained from impure syrups having a fucose content of more than 45% of DS, especially when the content of critical impurities is within a range below specific critical values. Fucose proved to have a very strong salting-out effect on other sugars, such as arabinose and rhamnose. For this reason, it has been very difficult to prepare fucose crystals with a high purity in the state of the art.